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The effect of iron oxide nanoparticles on Bacillus subtilis biofilm, growth and viability
Accepted Manuscript Title: The effect of iron oxide nanoparticles on Bacillus subtilis biofilm, growth and viability Authors: Dinali Ranmadugala, Alireza Ebrahiminezhad, Merilyn Manley-Harris, Younes Ghasemi, Aydin Berenjian PII: DOI: Reference:
S1359-5113(17)30603-7 http://dx.doi.org/doi:10.1016/j.procbio.2017.07.003 PRBI 11091
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
12-4-2017 26-6-2017 10-7-2017
Please cite this article as: Ranmadugala Dinali, Ebrahiminezhad Alireza, ManleyHarris Merilyn, Ghasemi Younes, Berenjian Aydin.The effect of iron oxide nanoparticles on Bacillus subtilis biofilm, growth and viability.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2017.07.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: The effect of iron oxide nanoparticles on Bacillus subtilis biofilm, growth and viability
Authors: Dinali Ranmadugalaa,b, Alireza Ebrahiminezhadc,d*, Merilyn Manley-Harrisa, Younes Ghasemid and Aydin Berenjian a*
Authors Affiliation: a
Faculty of Science & Engineering, University of Waikato, Hamilton, New Zealand;
b
National Aquatic Resources Research and Development Agency, Colombo 15, Sri Lanka;
c
Department of Medical Biotechnology, School of Medicine, and Noncommunicable
Diseases Research Centre, Fasa University of Medical Sciences, Fasa, Iran; d
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz,
Iran
Corresponding authors:
Dr. Aydin Berenjian Email: [email protected]
Dr. Alireza Ebrahiminezhad Email: [email protected]
Graphical abstract
Highlights
Superparamagnetic iron oxide (naked IONs) and 3-aminopropyltriethoxy silane coated IONs (APTES@IONs) were successfully prepared and characterised.
APTES@IONs found to be effective in reducing biofilm biomass without affecting the grwoth and cell viability of B. subtilis.
Naked IONs at higher concentrations significantly affected the viability of B. subtilis without significant reduction in biofilm biomass.
Abstract Bacillus subtilis is one of the key microorganisms for the industrial production of many value added products. The fermentation of this bacterium is associated with biofilm formation that results in major process and operational issues. The study of the effect of magnetic
nanoparticles on the bacterial cells can immensely contribute to designing intensified bioprocess methods. Present study was aimed at understanding the effect of superparamagnetic iron oxide (naked IONs) and 3-aminopropyltriethoxy silane coated IONs (IONs@APTES) on biofilm formation, growth and viability of Bacillus subtilis. Both IONs were successfully prepared and characterised by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction patterns (XRD) and vibrating sample magnetometry (VSM). IONs@APTES at 100µg/mL were found to significantly reduce the total biofilm biomass without affecting the cell viability compared with the behavior of naked IONS, which significantly affected the viability of B. subtilis at high concentrations without any significant redution in biofilm biomass. This suggests an active role of IONs@APTES in eliminating the biofilm formation as compared to the naked IONs. This work suggests the possibility of using IONs@APTES coated bacteria for a range of applications for future industrial fermentation with Bacillus subtilis.
Keywords Bacillus subtilis; biofilm; magnetic nanoparticles; fermentation
Introduction Bacillus subtilis is considered the key microorganism for industrial production of a wide range of value added compounds including vitamin K[1, 2], nattokinase [3] and surfactin [4]. One of the major problems with the use of B. subtilis in industrial fermentation is the formation of biofilms by this bacterium [5] which leads to many process and operational complications. Most importantly, mass transfer in biofilms is extremely limited and can lead to decreased metabolic activity [6]. Within the biofilm, bacteria also experience severe stress due to high cell density, limitation in nutrients,
accumulation of metabolites and high osmolarity [7]. In industrial settings, biofilm formation leads to costly periodic cleaning [8] and corrosion of equipment due to the protective nature, high survival competence [9] and extracellular enzyme production by biofilm bacteria. Importantly, endospore producing biofilm genera like Bacillus can become a significant source of steady contamination. In good manufacturing practice, countermeasures against biofilms are important to improve the process performance, product quality and quantity and also to reduce the incidents of costly cleaning. Controlling biofilm formation would be advantageous since it would address many process complications at the industrial scale [5]. Currently, there is intense scientific interest in nanomaterials research. While a variety of materials, such as proteins, polysaccharides and synthetic polymers, are used to prepare nanomaterials [10], iron oxide nanoparticles (IONs), with their unique magnetic properties, high surface/volume ratio and superior biocompatibility, show great promise for applications in bioprocesses [11-17]. The most commonly used IONs which display strong magnetic properties are magnetite (Fe3O4) nanoparticles [11, 12, 15, 18]. IONs have positively affected the biosynthesis of certain important products/metabolites such as menaquinone-7 by B. subtilis [13]. Further, IONs have been tested to reduce the number of downstream processing steps involved with the reuse of cells decorated with IONs in B. subtilis fermentation [13]. High cell capture efficiencies were recorded when the concentration of IONs were greater than 100μg/L [13]. However, according to Ebrahiminezhad and co-workers, attachment of IONs to bacterial surface has resulted in a profound growth inhibition at the end of fermentation [13]. Toxic effects of naked IONs on several cell lines have been reported through the generation of reactive oxygen species [17, 19]. For a particular cell line, therefore, IONs can be growth promoting or cytotoxic, mainly depending
upon the concentration [13, 19-21] and size [22] of the IONs. Biocompatible coatings can make IONs more biocompatible and surface active when compared to naked IONs [20, 23]. Therefore, the present study aimed to synthesize two types of IONs, naked and APTES coated, and evaluate their effects on B. subtilis biofilm formation, growth and viability in order to open up a new domain for designing intensified bioprocess methods for industrial production of a wide range of value added compounds in the near future.
Materials and methods
Synthesis of naked and APTES coated iron oxide nanoparticles
IONs were synthesized by co-precipitation of ferric and ferrous ions with ammonium hydroxide under a nitrogen atmosphere as described previously [11, 13, 18]. Briefly, FeSO4.7H2O (0.74g) and FeCl3.6H2O (1.17g) were dissolved in distilled water (50 mL) and the solution was vigorously stirred at 70°C under a nitrogen atmosphere for 1 h. Thereafter, 5mL of ammonium hydroxide solution was injected rapidly into the mixture while stirring continued for another 1 h. The resultant black precipitate was separated, washed with boiled distilled water, oven dried at 50°C overnight and stored in nitrogen. APTES coating was carried out as described previously [20]. Naked IONs (0.7 g) were dissolved in ethanol: water (25 mL, 1:1 v/v), and sonicated (10 min) to achieve a uniform dispersion. APTES (2.8 mL) was injected into the particle dispersion under a nitrogen atmosphere, while the temperature of the water bath was maintained at 40°C.
The reaction was continued (2 h, 40°C) with stirring. The resultant particles were washed with absolute ethanol and deionised water and oven dried at 50 °C overnight.
Characterization of naked and APTES coated iron oxide nanoparticles Particle size distribution and morphology were studied using transmission electron microscopy (TEM). Images of IONs and APTES coated IONs were taken on a Philips, CM 10; HT 100 Kv TEM, Philips Electron Optics, Eindhoven, Netherlands. Fourier transform infrared (FTIR) spectra were obtained using a Bruker, Vertex 70, FT-IR spectrometer Bruker, Kassel, Germany, in the range of 4000 to 400cm-1. For FTIR analysis, KBr pellets containing naked IONs and IONs@APTES were prepared. XRay powder diffraction patterns were obtained using a Siemens D5000 Vibrating sample magnetometer with 2-Theta ranging between 20° and 90°; magnetization measurements were conducted at room temperature.
Microorganism inoculum preparation B. subtilis (ATCC 6633) cells were cultured on nutrient agar plates (37°C, 2 days). A spore solution was prepared by suspending the cells in a 0.9% (w/v) sodium chloride solution and heating (30 min, 80°C) to inactivate the vegetative cells and produce spores. The spore solution was obtained after removing the cell debris by centrifugation at 3,000 rpm.
Crystal violet staining method (CVSM) and pellicle assay In order to test the effect of IONs@APTESs and naked IONs on B. subtilis biofilm formation, spores were grown up to 0.5 McFarland standard (OD 600 of 0.1) in LuriaBertani (LB) medium (2% w/v). The cell suspension was diluted 1:20 with fresh
media and incubated with shaking (37˚C, 120 rpm) with no IONS (control) and in the presence of varying concentrations of IONs@APTESs (100 to 400 µg/mL) as well as naked IONs in 30 mL glass vials. After incubation (60 h), the solutions were decanted and the vials were washed several times with distilled water. Biofilms were stained with crystal violet solution (0.05% w/v, 20 min). After staining the biofilms, the vials were gently rinsed with water and dried. The resulting stained biofilms were immersed in 30% aqueous acetic acid (20 min) to extract the pigments. One hundred and twentyfive microliters from each well was transferred to a new microtiter plate and the level of the crystal violet present in the destaining solution was measured at 550 nm using 30% aqueous acetic acid as a blank using a Multiskan TM Go microplate spectrophotometer (Thermoscientific). The mass of B. subtilis pellicles was ascertained by harvesting and drying in an oven (40˚C. 5 h); the dry weight was taken using a 5-figure balance.
Growth and viability of B. subtilis ATCC6633 The effects of IONs@APTES and naked IONs on the growth of B. subtilis cells were tested by the microdilution method according to the protocol of the Clinical and Laboratory Standards Institute [24-26]. A cell viability assay was also conducted by using LIVE/DEAD BacLight assay kit L7012 (Invitrogen™, Molecular Probes Inc.). Scanning electron microscopy (SEM) analysis Bacterial cultures were grown (60 h) in the absence of nanoparticles and in the presence of varying concentrations of IONs@APTES and naked IONs. Bacterial smears were prepared by placing a 10µl drop of cell suspension on a cover slip and heat drying by passing through the flame of a Bunsen burner followed by fixing with 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer at room temperature for 45 minutes (1L of 0.1M sodium cacodylate
buffer was prepared by dissolving 21.41g of sodium cacodylate in 900mL of distilled water followed by the addition of 8mL of 1N HCL and making up the volume to 1L). Coverslips were rinsed with normal saline four times over 30 min. Dehydration of the cells were done using a series of alcohol concentrations (50%, 75%, 95%) for 1 hour in each solution followed by four changes in absolute ethanol for 20 minutes. Specimens were subjected to critical point drying (Poloron) and the samples were mounted on aluminium stubs and coated with platinum before examination with a Scanning Electron Microscope (Hitachi S-4700).
Confocal Laser Scanning Microscopy (CLSM) analysis Biofilms were grown as described previously in LB in the presence of 100µg/mL of IONs@APTES and in the absence of nanoparticles for 60 hrs. Thereafter, biofilms were washed twice with 0.9% NaCl and were stained with live/dead staining. Stained biofilms were gently rinsed with 0.9% NaCl. Two independent biofilms were prepared for each condition. Biofilms were observed using a 60x oil immersion objective (60x/1.35 O) and images were acquired in an Olympus FluoView FV1000 (Olympus, Lisboa, Portugal) confocal laser microscope. Maximum biofilm thickness was determined after calculating the thickness of 20 different regions from biofilms grown under each condition as described before [27].
Statistical analysis Statistical significance was determined by analysis of variance (ANOVA) tests using IBM SPSS statistics. Data are the mean + standard error of replicates. Mean values were considered significantly different at p < 0.05.
Results and discussion Characterization of naked and APTES coated iron oxide nanoparticles TEM analysis of both naked and IONs@APTES revealed the production of fairly uniform spherical particles. The FTIR spectra of naked IONs and IONs@APTES are presented in Fig. 2. The Fe-O characteristic peaks of IONs@APTES appeared at about 637.2 cm-1 and for naked IONs at 644.52 cm-1. In IONs@APTES, the Si-O bond stretching vibration appeared at 1032.64 cm-1 [28]. Stretching vibrations of the OH groups appear at ~ 3400 cm-1 as a broad, characteristic peak, O-H bending results in a peak at 1630 cm-1 [12, 24, 25, 28, 29]. The peaks at 2910 and 2850 cm–1 correspond to the asymmetric and symmetric –C-H stretching vibrations and are indicative of the presence of aliphatic –CH2 groups. These results are in agreement that the IONs have been successfully coated with APTES. X-ray powder diffraction patterns of the nanoparticles were in agreement with characteristic features of the magnetite having intensity peaks at 2θ degrees of 30 °, 35.5°, 43°, 54°, 57° and 63° . The presence of intensity peaks at 2θ degrees of 30°, 35.5°, 43°, 54°, 57° and 63° in the X-ray powder diffraction pattern of IONs@APTES revealed that surface coating with APTES did not affect the crystal structure of magnetite (Fig. 3). An increase in peak intensities were seen after functionalization with APTES (Fig.3). The crystal size was calculated to be 17 nm by using Scherrer calculator toll on the PANalytical X'Pert HighScore (Produced by PAN alytical B.V., Almelo, the Netherlands, version 1.0d). Saturation magnetization analysis results are presented in Fig. 4. No hysteresis was seen and magnetization curves were completely reversible exhibiting the superparamagnetic behavior of the particles. The saturation magnetization (Ms) values were found to be 60 e.m.u/g for both naked IONs and IONs@APTES.
Effect of APTES coated IONs and naked IONs on biofilm formation by B. subtilis Crystal violet staining method (CVSM) The effect of the nanoparticles on biofilm formation is illustrated in Fig. 5. The analysis of variance test showed IONs@APTES reduced the surface-adherent biofilm biomass (p<0.05) and that this effect was generally concentration dependent, resulting in 13.93%, 9.76% and 7.39% reduction in the presence of 100, 200 and 300 µg/mL IONs@APTES, respectively. Dunnet’s multiple comparisons test show biofilm formation in the presence of 100, 200 and 300 µg/mL IONs@APTES are significantly lower than the control samples, which lacked IONs (p< 0.05). In contrast, treatment with similar concentrations of naked IONs showed no significant effect on reducing the biofilm formation even at high concentrations of naked IONs (300-400 µg/mL) (p>0.05). Similar observations have been reported previously [3032] but with a significant reduction in biofilm growth at higher concentration of naked IONs. Thukkaram et al. [31] showed IONs in a concentration range of 10 to 100 µg/mL were able to reduce biofilm growth by S. aureus, E.coli and P. aeruginosa. Similarly, Taylor and Webster [32] have reported a decrease in S. epidermidis numbers in a biofilm in the presence of 100 µg/mL of IONs. It has been suggested that the antibacterial effect of IONs may be either due to the production of reactive oxygen species or membrane damage due to electrostatic interactions or the small size of nanoparticles[32].
Pellicle assay and analysis Measurement of biofilms by the crystal violet staining method requires at least 24 to 48 h-cultures and estimates the adherent biofilm biomass stained after washing to
remove non-adherent cells. As the washing steps can significantly affect the final results, the total biofilm biomass was also determined by a pellicle assay. IONs@APTES showed a significant reduction in biofilm formation compared to naked IONs (Fig. 6). Post hoc analysis by Dunnet’s multiple comparison test showed that biofilm formation in the presence of 100 and 200 µg/mL IONs@APTES were significantly lower than the control (p< 0.05). The pellicle assay was consistent with the CVSM showing that 100 and 200 µg/mL IONs@APTES exhibited comparably significant reduction in biofilm biomass measured by both methods (p< 0.05). Since there was little difference between the 100 and 200 µg/mL IONs@APTES assays, the lower concentration is preferred for biofilm control. The exact mechanism by which IONs@APTES inhibit the adherence of B. subtilis to surfaces and reduce pellicle formation was not investigated in this study. However, bacterial attachment to any surface is related to surface charge of both substratum and bacteria [33]. Attachment of B. subtilis to IONs@APTES is mainly driven by the attractive electrostatic interaction between the positively charged amine functional groups on IONS@APTES and the negatively charged cell surface of bacteria. It can be speculated that the significant reduction of biofilm formation in the presence of IONS@APTES may be due to the attachment of IONs@APTES to the cell wall, thereby changing the surface charge and surface binding potential of cells. Further, once attached to bacterial cell surface, IONs@APTES can act as a physical barrier for the attachment of bacterial cells to other surfaces and initiation of biofilm formation. Naked IONs might have shown limited use in controlling biofilm formation as they are not well dispersed at higher concentrations; an external magnetic field has sometimes been used to target and penetrate IONs deep into the biofilm and concentrate them therein [34] in order to eliminate preformed biofilms. In this regard,
size and physicochemical characteristics of IONs have both played an important role in their diffusion through biofilms [34] and disruption of biofilms through the bactericidal effect exerted by the IONs. In contrast, APTES coating seems to stabilize the IONs and maintain them in a dispersed state while specifically interacting with the bacterium and changing the adhesion pattern and subsequent pellicle formation of B. subtilis.
Effect of APTES coated IONs and naked IONs on growth and viability of B. subtilis In successful cell immobilization, the immobilized cells need to express their activities such as growth and metabolic activities. To this end, we further measured the growth/cell density and viability of B. subtilis in the presence of varying concentrations of IONs@APTES, naked IONs and in the absence of these nanoparticles.
Bacterial growth IONs@APTES showed more growth promoting effect than the naked IONs. With increasing concentrations of IONs@APTES an increase of growth/population density was seen after 60 hours as shown in Fig. 7. Bacteria surviving and colonizing on surfaces functionalised with APTES have been reported previously by Lee et al. [35]. It is possible that the binding of IONs@APTES to B. subtilis has changed the membrane permeability and has facilitated mass transfer and nutrient absorption in a concentration dependent manner. Supporting observations have been reported by Ansari et al. [36] in which coating of bacterial cells with magnetic nanoparticles resulted in higher dibenzthiophene activity. It was suggested that this was due to changes in cell membrane permeability which facilitated the entry and exit of reactant and product [36].
The increase in cell density in the presence of varying concentrations of IONs@APTES showed a similar pattern to increase in biofilm biomass. However, the cell density was not significantly less than the control in the presence of 100 and 200 µg/mL of IONs@APTES although the biofilm biomass was significantly less. The results support the idea that IONs@APTES compromise surface adherence of B. subtilis to glass surface and pellicle formation by altering the adhesion pattern of the bacterium without affecting microbial growth. Naked IONs showed a growth promoting effect up to 200 µg/mL. Similar observations have been reported previously where IONs have induced growth by acting as an endogenous source of iron for bacteria, and this effect is dependent on the particle size of IONs [22] as well as the concentration of IONs [20]. The population density of B. subtilis is significantly reduced in the presence of high concentrations of naked IONs (300 and 400 µg/mL) (p< 0.05); however, the reduction in biofilm biomass was not significantly different from the control, even in the presence of higher concentrations of naked IONs, although the growth/cell density was significantly less. As biofilm bacteria as well as planktonic bacteria contribute to the optical density value, it can be speculated that the significant reduction in optical density in the presence of high concentration of naked IONs may be due to the significant antibacterial effect of naked IONs on planktonic bacteria at high concentrations. As naked IONs are not well dispersed at higher concentrations, response of biofilm bacteria and planktonic bacteria to IONs can be different. A reduction of B. subtilis growth in the presence of naked IONs with an average size of 11nm, in a concentration and time dependent manner have been demonstrated by Ebrahiminezhad and co-workers [13]. Further, Arakha and co-workers reported a reduction in B. subtilis growth with increasing concentration of naked IONs with an estimated particle size of approximately 11+/- 5nm; however, the growth inhibition was insignificant for the concentrations of naked IONs studied [37]. Growth
inhibitory effect of naked IONs on many other bacterial species, such as S. aureus, E. coli, L. monocytogenes and P. aeruginosa, in a concentration dependent manner, have also been reported [12, 13, 22, 38, 39]. The growth inhibition has been attributed to the production of reactive oxygen species (ROS) resulting in oxidative stress and damage to cell membrane, protein and DNA [21]. Therefore, in comparison to naked IONs, IONs@APTES present a better option for immobilisation of B. subtilis and controlling biofilm formation while maintaining bacterial growth.
Bacterial cell viability Optical density values do not distinguish between dead and live cells. Therefore we further investigated the bacterial cell viability in the presence of naked IONs and IONs@APTES using a LIVE/DEAD assay. In industrial fermentation biofilm control and growth and viability of the bacterium are equally important; however, previous studies with naked IONS showed that the reduction in biofilm is due to their antibacterial potential [30-32, 40]. As shown in Fig. 8, although there was a significant reduction in biofilm formation in the presence of 100 and 200µg/mL of IONs@APTES, none of the IONs@APTES concentrations significantly affected the total cell viability of B. subtilis (p> 0.05). Similarly, Gottenbos et al. reported that positively charged surfaces of biomaterial do not affect surface growth nor exert antimicrobial effect on adhering gram positive bacteria [41]. The results have been ascribed to the thick and rigid peptidoglycan layer of gram positive bacteria where it prevents/reduces extensive contact of ammonium groups with the membrane, even under conditions of electrostatic attractions [41]. Therefore it is speculated that the reduction in biofilm biomass in the presence of low concentrations of IONs@APTES may be due to the change in surface binding potential or shielding effect of
IONs@APTES leading to less adherence and pellicle formation of B. subtilis. In contrast, total cell viability of B. subtilis was significantly less in the presence of high concentrations of naked IONs (300 and 400 µg/mL) (p<0.05), which may be due to the toxic effect exerted by naked IONs. Similarly, Arakha and co-workers reported a 30% reduction in the viability of B. subtilis in the presence of 50µM of naked IONs with an estimated particle size of approximately 11+/- 5nm[37]. Toxic effect of naked IONs on cell lines in a concentration dependent manner have also been reported by several other studies [13, 21, 34, 38, 39].
Microscopic observations In addition to the information provided by the in vitro viability assay, we further evaluated the interaction of B. subtilis (ATCC6633) with naked and IONs@APTES using a scanning electron microscope (SEM). Both naked IONs and IONs@APTES were attached to B. subtilis cells as shown in Fig. 9; however, B. subtilis cells were more densely coated with IONs@APTES (Figure 9a) in comparison to naked IONs (Figure 9b). B. subtilis cells growing the absence of nanoparticles are represented by Figure 9c. According to the results, IONs@APTES seem to have provided a perfect platform for the attachment of B. subtilis. In accordance with our study, several other studies show that nanoparticles with positive surface potential would provide unique advantage of attracting bacteria [16, 42, 43]. Interestingly, morphological changes, such as increased cell length in some of the cells, were seen in cultures grown in the presence of IONs@APTES and naked IONs as well as in the absence of nanoparticles (Figures 9d & e). These morphs can be readily differentiated from the ancestral short rods of B. subtilis. Change in cell morphology is a possibility in bacteria-nanoparticle interaction. For example, Chatterjee [21] reported an abrupt increase in cell length of E. coli in the presence of
iron oxide nanoparticles. However, in this study, the presence of filamentous morphs, even in the absence of nanoparticles, eliminates the possibility of filamentation due to nanoparticle-bacterial interaction and suggests that the presence of filamentous morphs of B. subtilis may be due to physiological changes in some cells within the biofilm matrix. Non-motile, long cell chains have been reported previously in B. subtilis biofilms [44] and in many other bacteria including B. cereus, E. coli, L monocytogenes and Salmonella spp. [45]. Cell elongation/changes in morphology has been described as a survival mechanism to confer protection against stressful environmental situations [46, 47]. These morphotypes are regarded as viable since filamentation is a reversible stress response and the cells are able to divide and form new normal cells upon removal of the stress conditions [45]. According to scanning electron micrographs, IONs@APTES were seen concentrated within biofilms without the need of an external magnetic field (Figure 9f). Nanoparticles were also seen attached to the extracellular polymeric matrix which forms a complex network in biofilms (Figure 9f). As the majority of bacteria have negatively charged biofilm matrixes [48], positively charged IONs@APTES with high surface/volume ratio, which also disperse easily in the aqueous medium, can readily penetrate and bind to the biofilm matrix. Previous studies have also demonstrated that positively charged particles would more easily penetrate the negatively charged biofilms [34]. Apart from the charge, the size of IONs can also impact their diffusion through biofilms[34]. IONs@APTES significantly reduced the biofilm density as compared to the untreated cells (Figure 10). Despite the fact that IONs@APTES did not effectively kill biofilm bacteria in comparison to the untreated cells, CLSM analysis revealed that biofilm maximum depth, which is an indirect measure of the amount of B. subtilis biofilm biomass in the presence of
IONs@APTES, is 28.66 µm in depth which was significantly different from the depth of the biofilm in the absence of nanoparticles which was 44.60 µm (p<0.05) (Figure 11). Taken together, the results support the idea that IONs@APTES, with positive surface potential, hold great promise in controlling B. subtilis biofilm formation without compromising bacterial growth and viability.
Conclusions Growth and biofilm formation of B. subtilis has a profound effect on many bacterial bioprocesses. The present study was focused on immobilization of B. subtilis with naked and APTES coated IONs and evaluation of their effects on B. subtilis biofilm formation, growth and viability. In comparison to naked IONS, IONs@APTES offered a high density of surface functional groups of –NH3+ to IONs, promoting strong electrostatic interaction with negatively charged sites on the cell membranes allowing good decoration of cells. IONs@APTES have been demonstrated to be a promising approach in control of biofilm formation without the loss of B. subtilis cell viability compared to naked IONs. APTES coated magnetic nanoparticles, at appropriate concentrations, offer a considerable advantage for biofilm control while maintaining the growth and viability of bacterial cells when designing the next generation of bioprocesses intensification methods. Because of the overall significance of biofilm control, it is necessary to further study the spatial and temporal patterns of biofilm formation in the presence of IONs@APTES and the molecular basis of these interactions using an appropriate platform for biofilm experiments.
Ethical approval This study does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements This investigation was financially supported by The University of Waikato, New Zealand. The support given by Ms. Helen Turner, Dr. Barry O’Brian, Dr. Judith Burrows and Dr. Lisa Lee is highly appreciated. A special thanks to Dr. Andrew J. Spheres for the valuable suggestions in biofilm studies.
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Figure captions
Fig. 1 Transmission electron micrographs of a) naked IONs and b) IONs@APTES
Fig. 2 Fourier transform infrared spectroscopy (FTIR) spectra of a) naked IONs and b) IONs@APTES
Fig. 3 X—ray powder diffraction patterns of a) naked IONS and b) IONs@APTES
Fig. 4 Vibrating sample magnetometer (VSM) diagrams of a) naked IONs and b) IONs@APTES
Fig. 5 a crystal violet quantification of B. subtilis biofilm in the presence of IONs@APTES and naked IONs, b crystal violet stained biofilms using different IONs@APTES concentration (from left to right 0-400 µg/mL) and c crystal violet stained biofilms using different naked IONS concentrations (from left to right 0-400 µg/mL). Results are expressed as the absorbance measured at 550nm. Data are the means + standard error of 3 replicates. * p<0.05
Fig. 6 Quantification of B. subtilis biofilm pellicle biomass in the presence of IONs@APTES and naked IONs.The formation of a biofilm was measured after 60 hours using the pellicle biomass assay. Results are expressed as dry weight (g). Data are the means + standard error of 3 replicates. * p<0.05
Fig. 7 Population density of B. subtilis in the presence of IONs@APTES and naked IONs. Population density was measured by reading OD values at 600nm after 60 hours. Data are the means + standard error of 3 replicates. * p<0.05
Fig. 8 Viability of B. subtilis in the presence of IONs@APTES and naked IONs. Viability was assessed using LIVE/DEAD BacLight assay kit L7012 after 60 hours. Data are the means + standard error of 3 replicates. * p<0.05
Fig. 9 Scanning electron micrographs of B. subtilis growing in the presence of a IONs@APTES, b naked IONs, c control conditions, and filamentous morphs of B. subtilis growing d in the presence of IONs@APTES, e in the absence of nanoparticles, f B. subtilis biofilm matrix
Fig. 10 CLSM images of 60-h B. subtilis biofilm in the presence of 100µg/mL IONs@APTES (a and b) and in the absence of nanoparticles (c and d). Biofilms were stained with live/dead. Live cells are stained in green (a and c) dead cells stained in red (b and d)
Fig. 11 Biofilm maximum depth average obtained by CLSM for B. subtilis biofilm in the absence of nanoparticles and in the presence of 100 µg/mL IONs@APTES after 60 hours. *statistically different from control (untreated cells) (p< 0. 05)
S1359-5113(17)30603-7 http://dx.doi.org/doi:10.1016/j.procbio.2017.07.003 PRBI 11091
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
12-4-2017 26-6-2017 10-7-2017
Please cite this article as: Ranmadugala Dinali, Ebrahiminezhad Alireza, ManleyHarris Merilyn, Ghasemi Younes, Berenjian Aydin.The effect of iron oxide nanoparticles on Bacillus subtilis biofilm, growth and viability.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2017.07.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: The effect of iron oxide nanoparticles on Bacillus subtilis biofilm, growth and viability
Authors: Dinali Ranmadugalaa,b, Alireza Ebrahiminezhadc,d*, Merilyn Manley-Harrisa, Younes Ghasemid and Aydin Berenjian a*
Authors Affiliation: a
Faculty of Science & Engineering, University of Waikato, Hamilton, New Zealand;
b
National Aquatic Resources Research and Development Agency, Colombo 15, Sri Lanka;
c
Department of Medical Biotechnology, School of Medicine, and Noncommunicable
Diseases Research Centre, Fasa University of Medical Sciences, Fasa, Iran; d
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz,
Iran
Corresponding authors:
Dr. Aydin Berenjian Email: [email protected]
Dr. Alireza Ebrahiminezhad Email: [email protected]
Graphical abstract
Highlights
Superparamagnetic iron oxide (naked IONs) and 3-aminopropyltriethoxy silane coated IONs (APTES@IONs) were successfully prepared and characterised.
APTES@IONs found to be effective in reducing biofilm biomass without affecting the grwoth and cell viability of B. subtilis.
Naked IONs at higher concentrations significantly affected the viability of B. subtilis without significant reduction in biofilm biomass.
Abstract Bacillus subtilis is one of the key microorganisms for the industrial production of many value added products. The fermentation of this bacterium is associated with biofilm formation that results in major process and operational issues. The study of the effect of magnetic
nanoparticles on the bacterial cells can immensely contribute to designing intensified bioprocess methods. Present study was aimed at understanding the effect of superparamagnetic iron oxide (naked IONs) and 3-aminopropyltriethoxy silane coated IONs (IONs@APTES) on biofilm formation, growth and viability of Bacillus subtilis. Both IONs were successfully prepared and characterised by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction patterns (XRD) and vibrating sample magnetometry (VSM). IONs@APTES at 100µg/mL were found to significantly reduce the total biofilm biomass without affecting the cell viability compared with the behavior of naked IONS, which significantly affected the viability of B. subtilis at high concentrations without any significant redution in biofilm biomass. This suggests an active role of IONs@APTES in eliminating the biofilm formation as compared to the naked IONs. This work suggests the possibility of using IONs@APTES coated bacteria for a range of applications for future industrial fermentation with Bacillus subtilis.
Keywords Bacillus subtilis; biofilm; magnetic nanoparticles; fermentation
Introduction Bacillus subtilis is considered the key microorganism for industrial production of a wide range of value added compounds including vitamin K[1, 2], nattokinase [3] and surfactin [4]. One of the major problems with the use of B. subtilis in industrial fermentation is the formation of biofilms by this bacterium [5] which leads to many process and operational complications. Most importantly, mass transfer in biofilms is extremely limited and can lead to decreased metabolic activity [6]. Within the biofilm, bacteria also experience severe stress due to high cell density, limitation in nutrients,
accumulation of metabolites and high osmolarity [7]. In industrial settings, biofilm formation leads to costly periodic cleaning [8] and corrosion of equipment due to the protective nature, high survival competence [9] and extracellular enzyme production by biofilm bacteria. Importantly, endospore producing biofilm genera like Bacillus can become a significant source of steady contamination. In good manufacturing practice, countermeasures against biofilms are important to improve the process performance, product quality and quantity and also to reduce the incidents of costly cleaning. Controlling biofilm formation would be advantageous since it would address many process complications at the industrial scale [5]. Currently, there is intense scientific interest in nanomaterials research. While a variety of materials, such as proteins, polysaccharides and synthetic polymers, are used to prepare nanomaterials [10], iron oxide nanoparticles (IONs), with their unique magnetic properties, high surface/volume ratio and superior biocompatibility, show great promise for applications in bioprocesses [11-17]. The most commonly used IONs which display strong magnetic properties are magnetite (Fe3O4) nanoparticles [11, 12, 15, 18]. IONs have positively affected the biosynthesis of certain important products/metabolites such as menaquinone-7 by B. subtilis [13]. Further, IONs have been tested to reduce the number of downstream processing steps involved with the reuse of cells decorated with IONs in B. subtilis fermentation [13]. High cell capture efficiencies were recorded when the concentration of IONs were greater than 100μg/L [13]. However, according to Ebrahiminezhad and co-workers, attachment of IONs to bacterial surface has resulted in a profound growth inhibition at the end of fermentation [13]. Toxic effects of naked IONs on several cell lines have been reported through the generation of reactive oxygen species [17, 19]. For a particular cell line, therefore, IONs can be growth promoting or cytotoxic, mainly depending
upon the concentration [13, 19-21] and size [22] of the IONs. Biocompatible coatings can make IONs more biocompatible and surface active when compared to naked IONs [20, 23]. Therefore, the present study aimed to synthesize two types of IONs, naked and APTES coated, and evaluate their effects on B. subtilis biofilm formation, growth and viability in order to open up a new domain for designing intensified bioprocess methods for industrial production of a wide range of value added compounds in the near future.
Materials and methods
Synthesis of naked and APTES coated iron oxide nanoparticles
IONs were synthesized by co-precipitation of ferric and ferrous ions with ammonium hydroxide under a nitrogen atmosphere as described previously [11, 13, 18]. Briefly, FeSO4.7H2O (0.74g) and FeCl3.6H2O (1.17g) were dissolved in distilled water (50 mL) and the solution was vigorously stirred at 70°C under a nitrogen atmosphere for 1 h. Thereafter, 5mL of ammonium hydroxide solution was injected rapidly into the mixture while stirring continued for another 1 h. The resultant black precipitate was separated, washed with boiled distilled water, oven dried at 50°C overnight and stored in nitrogen. APTES coating was carried out as described previously [20]. Naked IONs (0.7 g) were dissolved in ethanol: water (25 mL, 1:1 v/v), and sonicated (10 min) to achieve a uniform dispersion. APTES (2.8 mL) was injected into the particle dispersion under a nitrogen atmosphere, while the temperature of the water bath was maintained at 40°C.
The reaction was continued (2 h, 40°C) with stirring. The resultant particles were washed with absolute ethanol and deionised water and oven dried at 50 °C overnight.
Characterization of naked and APTES coated iron oxide nanoparticles Particle size distribution and morphology were studied using transmission electron microscopy (TEM). Images of IONs and APTES coated IONs were taken on a Philips, CM 10; HT 100 Kv TEM, Philips Electron Optics, Eindhoven, Netherlands. Fourier transform infrared (FTIR) spectra were obtained using a Bruker, Vertex 70, FT-IR spectrometer Bruker, Kassel, Germany, in the range of 4000 to 400cm-1. For FTIR analysis, KBr pellets containing naked IONs and IONs@APTES were prepared. XRay powder diffraction patterns were obtained using a Siemens D5000 Vibrating sample magnetometer with 2-Theta ranging between 20° and 90°; magnetization measurements were conducted at room temperature.
Microorganism inoculum preparation B. subtilis (ATCC 6633) cells were cultured on nutrient agar plates (37°C, 2 days). A spore solution was prepared by suspending the cells in a 0.9% (w/v) sodium chloride solution and heating (30 min, 80°C) to inactivate the vegetative cells and produce spores. The spore solution was obtained after removing the cell debris by centrifugation at 3,000 rpm.
Crystal violet staining method (CVSM) and pellicle assay In order to test the effect of IONs@APTESs and naked IONs on B. subtilis biofilm formation, spores were grown up to 0.5 McFarland standard (OD 600 of 0.1) in LuriaBertani (LB) medium (2% w/v). The cell suspension was diluted 1:20 with fresh
media and incubated with shaking (37˚C, 120 rpm) with no IONS (control) and in the presence of varying concentrations of IONs@APTESs (100 to 400 µg/mL) as well as naked IONs in 30 mL glass vials. After incubation (60 h), the solutions were decanted and the vials were washed several times with distilled water. Biofilms were stained with crystal violet solution (0.05% w/v, 20 min). After staining the biofilms, the vials were gently rinsed with water and dried. The resulting stained biofilms were immersed in 30% aqueous acetic acid (20 min) to extract the pigments. One hundred and twentyfive microliters from each well was transferred to a new microtiter plate and the level of the crystal violet present in the destaining solution was measured at 550 nm using 30% aqueous acetic acid as a blank using a Multiskan TM Go microplate spectrophotometer (Thermoscientific). The mass of B. subtilis pellicles was ascertained by harvesting and drying in an oven (40˚C. 5 h); the dry weight was taken using a 5-figure balance.
Growth and viability of B. subtilis ATCC6633 The effects of IONs@APTES and naked IONs on the growth of B. subtilis cells were tested by the microdilution method according to the protocol of the Clinical and Laboratory Standards Institute [24-26]. A cell viability assay was also conducted by using LIVE/DEAD BacLight assay kit L7012 (Invitrogen™, Molecular Probes Inc.). Scanning electron microscopy (SEM) analysis Bacterial cultures were grown (60 h) in the absence of nanoparticles and in the presence of varying concentrations of IONs@APTES and naked IONs. Bacterial smears were prepared by placing a 10µl drop of cell suspension on a cover slip and heat drying by passing through the flame of a Bunsen burner followed by fixing with 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer at room temperature for 45 minutes (1L of 0.1M sodium cacodylate
buffer was prepared by dissolving 21.41g of sodium cacodylate in 900mL of distilled water followed by the addition of 8mL of 1N HCL and making up the volume to 1L). Coverslips were rinsed with normal saline four times over 30 min. Dehydration of the cells were done using a series of alcohol concentrations (50%, 75%, 95%) for 1 hour in each solution followed by four changes in absolute ethanol for 20 minutes. Specimens were subjected to critical point drying (Poloron) and the samples were mounted on aluminium stubs and coated with platinum before examination with a Scanning Electron Microscope (Hitachi S-4700).
Confocal Laser Scanning Microscopy (CLSM) analysis Biofilms were grown as described previously in LB in the presence of 100µg/mL of IONs@APTES and in the absence of nanoparticles for 60 hrs. Thereafter, biofilms were washed twice with 0.9% NaCl and were stained with live/dead staining. Stained biofilms were gently rinsed with 0.9% NaCl. Two independent biofilms were prepared for each condition. Biofilms were observed using a 60x oil immersion objective (60x/1.35 O) and images were acquired in an Olympus FluoView FV1000 (Olympus, Lisboa, Portugal) confocal laser microscope. Maximum biofilm thickness was determined after calculating the thickness of 20 different regions from biofilms grown under each condition as described before [27].
Statistical analysis Statistical significance was determined by analysis of variance (ANOVA) tests using IBM SPSS statistics. Data are the mean + standard error of replicates. Mean values were considered significantly different at p < 0.05.
Results and discussion Characterization of naked and APTES coated iron oxide nanoparticles TEM analysis of both naked and IONs@APTES revealed the production of fairly uniform spherical particles. The FTIR spectra of naked IONs and IONs@APTES are presented in Fig. 2. The Fe-O characteristic peaks of IONs@APTES appeared at about 637.2 cm-1 and for naked IONs at 644.52 cm-1. In IONs@APTES, the Si-O bond stretching vibration appeared at 1032.64 cm-1 [28]. Stretching vibrations of the OH groups appear at ~ 3400 cm-1 as a broad, characteristic peak, O-H bending results in a peak at 1630 cm-1 [12, 24, 25, 28, 29]. The peaks at 2910 and 2850 cm–1 correspond to the asymmetric and symmetric –C-H stretching vibrations and are indicative of the presence of aliphatic –CH2 groups. These results are in agreement that the IONs have been successfully coated with APTES. X-ray powder diffraction patterns of the nanoparticles were in agreement with characteristic features of the magnetite having intensity peaks at 2θ degrees of 30 °, 35.5°, 43°, 54°, 57° and 63° . The presence of intensity peaks at 2θ degrees of 30°, 35.5°, 43°, 54°, 57° and 63° in the X-ray powder diffraction pattern of IONs@APTES revealed that surface coating with APTES did not affect the crystal structure of magnetite (Fig. 3). An increase in peak intensities were seen after functionalization with APTES (Fig.3). The crystal size was calculated to be 17 nm by using Scherrer calculator toll on the PANalytical X'Pert HighScore (Produced by PAN alytical B.V., Almelo, the Netherlands, version 1.0d). Saturation magnetization analysis results are presented in Fig. 4. No hysteresis was seen and magnetization curves were completely reversible exhibiting the superparamagnetic behavior of the particles. The saturation magnetization (Ms) values were found to be 60 e.m.u/g for both naked IONs and IONs@APTES.
Effect of APTES coated IONs and naked IONs on biofilm formation by B. subtilis Crystal violet staining method (CVSM) The effect of the nanoparticles on biofilm formation is illustrated in Fig. 5. The analysis of variance test showed IONs@APTES reduced the surface-adherent biofilm biomass (p<0.05) and that this effect was generally concentration dependent, resulting in 13.93%, 9.76% and 7.39% reduction in the presence of 100, 200 and 300 µg/mL IONs@APTES, respectively. Dunnet’s multiple comparisons test show biofilm formation in the presence of 100, 200 and 300 µg/mL IONs@APTES are significantly lower than the control samples, which lacked IONs (p< 0.05). In contrast, treatment with similar concentrations of naked IONs showed no significant effect on reducing the biofilm formation even at high concentrations of naked IONs (300-400 µg/mL) (p>0.05). Similar observations have been reported previously [3032] but with a significant reduction in biofilm growth at higher concentration of naked IONs. Thukkaram et al. [31] showed IONs in a concentration range of 10 to 100 µg/mL were able to reduce biofilm growth by S. aureus, E.coli and P. aeruginosa. Similarly, Taylor and Webster [32] have reported a decrease in S. epidermidis numbers in a biofilm in the presence of 100 µg/mL of IONs. It has been suggested that the antibacterial effect of IONs may be either due to the production of reactive oxygen species or membrane damage due to electrostatic interactions or the small size of nanoparticles[32].
Pellicle assay and analysis Measurement of biofilms by the crystal violet staining method requires at least 24 to 48 h-cultures and estimates the adherent biofilm biomass stained after washing to
remove non-adherent cells. As the washing steps can significantly affect the final results, the total biofilm biomass was also determined by a pellicle assay. IONs@APTES showed a significant reduction in biofilm formation compared to naked IONs (Fig. 6). Post hoc analysis by Dunnet’s multiple comparison test showed that biofilm formation in the presence of 100 and 200 µg/mL IONs@APTES were significantly lower than the control (p< 0.05). The pellicle assay was consistent with the CVSM showing that 100 and 200 µg/mL IONs@APTES exhibited comparably significant reduction in biofilm biomass measured by both methods (p< 0.05). Since there was little difference between the 100 and 200 µg/mL IONs@APTES assays, the lower concentration is preferred for biofilm control. The exact mechanism by which IONs@APTES inhibit the adherence of B. subtilis to surfaces and reduce pellicle formation was not investigated in this study. However, bacterial attachment to any surface is related to surface charge of both substratum and bacteria [33]. Attachment of B. subtilis to IONs@APTES is mainly driven by the attractive electrostatic interaction between the positively charged amine functional groups on IONS@APTES and the negatively charged cell surface of bacteria. It can be speculated that the significant reduction of biofilm formation in the presence of IONS@APTES may be due to the attachment of IONs@APTES to the cell wall, thereby changing the surface charge and surface binding potential of cells. Further, once attached to bacterial cell surface, IONs@APTES can act as a physical barrier for the attachment of bacterial cells to other surfaces and initiation of biofilm formation. Naked IONs might have shown limited use in controlling biofilm formation as they are not well dispersed at higher concentrations; an external magnetic field has sometimes been used to target and penetrate IONs deep into the biofilm and concentrate them therein [34] in order to eliminate preformed biofilms. In this regard,
size and physicochemical characteristics of IONs have both played an important role in their diffusion through biofilms [34] and disruption of biofilms through the bactericidal effect exerted by the IONs. In contrast, APTES coating seems to stabilize the IONs and maintain them in a dispersed state while specifically interacting with the bacterium and changing the adhesion pattern and subsequent pellicle formation of B. subtilis.
Effect of APTES coated IONs and naked IONs on growth and viability of B. subtilis In successful cell immobilization, the immobilized cells need to express their activities such as growth and metabolic activities. To this end, we further measured the growth/cell density and viability of B. subtilis in the presence of varying concentrations of IONs@APTES, naked IONs and in the absence of these nanoparticles.
Bacterial growth IONs@APTES showed more growth promoting effect than the naked IONs. With increasing concentrations of IONs@APTES an increase of growth/population density was seen after 60 hours as shown in Fig. 7. Bacteria surviving and colonizing on surfaces functionalised with APTES have been reported previously by Lee et al. [35]. It is possible that the binding of IONs@APTES to B. subtilis has changed the membrane permeability and has facilitated mass transfer and nutrient absorption in a concentration dependent manner. Supporting observations have been reported by Ansari et al. [36] in which coating of bacterial cells with magnetic nanoparticles resulted in higher dibenzthiophene activity. It was suggested that this was due to changes in cell membrane permeability which facilitated the entry and exit of reactant and product [36].
The increase in cell density in the presence of varying concentrations of IONs@APTES showed a similar pattern to increase in biofilm biomass. However, the cell density was not significantly less than the control in the presence of 100 and 200 µg/mL of IONs@APTES although the biofilm biomass was significantly less. The results support the idea that IONs@APTES compromise surface adherence of B. subtilis to glass surface and pellicle formation by altering the adhesion pattern of the bacterium without affecting microbial growth. Naked IONs showed a growth promoting effect up to 200 µg/mL. Similar observations have been reported previously where IONs have induced growth by acting as an endogenous source of iron for bacteria, and this effect is dependent on the particle size of IONs [22] as well as the concentration of IONs [20]. The population density of B. subtilis is significantly reduced in the presence of high concentrations of naked IONs (300 and 400 µg/mL) (p< 0.05); however, the reduction in biofilm biomass was not significantly different from the control, even in the presence of higher concentrations of naked IONs, although the growth/cell density was significantly less. As biofilm bacteria as well as planktonic bacteria contribute to the optical density value, it can be speculated that the significant reduction in optical density in the presence of high concentration of naked IONs may be due to the significant antibacterial effect of naked IONs on planktonic bacteria at high concentrations. As naked IONs are not well dispersed at higher concentrations, response of biofilm bacteria and planktonic bacteria to IONs can be different. A reduction of B. subtilis growth in the presence of naked IONs with an average size of 11nm, in a concentration and time dependent manner have been demonstrated by Ebrahiminezhad and co-workers [13]. Further, Arakha and co-workers reported a reduction in B. subtilis growth with increasing concentration of naked IONs with an estimated particle size of approximately 11+/- 5nm; however, the growth inhibition was insignificant for the concentrations of naked IONs studied [37]. Growth
inhibitory effect of naked IONs on many other bacterial species, such as S. aureus, E. coli, L. monocytogenes and P. aeruginosa, in a concentration dependent manner, have also been reported [12, 13, 22, 38, 39]. The growth inhibition has been attributed to the production of reactive oxygen species (ROS) resulting in oxidative stress and damage to cell membrane, protein and DNA [21]. Therefore, in comparison to naked IONs, IONs@APTES present a better option for immobilisation of B. subtilis and controlling biofilm formation while maintaining bacterial growth.
Bacterial cell viability Optical density values do not distinguish between dead and live cells. Therefore we further investigated the bacterial cell viability in the presence of naked IONs and IONs@APTES using a LIVE/DEAD assay. In industrial fermentation biofilm control and growth and viability of the bacterium are equally important; however, previous studies with naked IONS showed that the reduction in biofilm is due to their antibacterial potential [30-32, 40]. As shown in Fig. 8, although there was a significant reduction in biofilm formation in the presence of 100 and 200µg/mL of IONs@APTES, none of the IONs@APTES concentrations significantly affected the total cell viability of B. subtilis (p> 0.05). Similarly, Gottenbos et al. reported that positively charged surfaces of biomaterial do not affect surface growth nor exert antimicrobial effect on adhering gram positive bacteria [41]. The results have been ascribed to the thick and rigid peptidoglycan layer of gram positive bacteria where it prevents/reduces extensive contact of ammonium groups with the membrane, even under conditions of electrostatic attractions [41]. Therefore it is speculated that the reduction in biofilm biomass in the presence of low concentrations of IONs@APTES may be due to the change in surface binding potential or shielding effect of
IONs@APTES leading to less adherence and pellicle formation of B. subtilis. In contrast, total cell viability of B. subtilis was significantly less in the presence of high concentrations of naked IONs (300 and 400 µg/mL) (p<0.05), which may be due to the toxic effect exerted by naked IONs. Similarly, Arakha and co-workers reported a 30% reduction in the viability of B. subtilis in the presence of 50µM of naked IONs with an estimated particle size of approximately 11+/- 5nm[37]. Toxic effect of naked IONs on cell lines in a concentration dependent manner have also been reported by several other studies [13, 21, 34, 38, 39].
Microscopic observations In addition to the information provided by the in vitro viability assay, we further evaluated the interaction of B. subtilis (ATCC6633) with naked and IONs@APTES using a scanning electron microscope (SEM). Both naked IONs and IONs@APTES were attached to B. subtilis cells as shown in Fig. 9; however, B. subtilis cells were more densely coated with IONs@APTES (Figure 9a) in comparison to naked IONs (Figure 9b). B. subtilis cells growing the absence of nanoparticles are represented by Figure 9c. According to the results, IONs@APTES seem to have provided a perfect platform for the attachment of B. subtilis. In accordance with our study, several other studies show that nanoparticles with positive surface potential would provide unique advantage of attracting bacteria [16, 42, 43]. Interestingly, morphological changes, such as increased cell length in some of the cells, were seen in cultures grown in the presence of IONs@APTES and naked IONs as well as in the absence of nanoparticles (Figures 9d & e). These morphs can be readily differentiated from the ancestral short rods of B. subtilis. Change in cell morphology is a possibility in bacteria-nanoparticle interaction. For example, Chatterjee [21] reported an abrupt increase in cell length of E. coli in the presence of
iron oxide nanoparticles. However, in this study, the presence of filamentous morphs, even in the absence of nanoparticles, eliminates the possibility of filamentation due to nanoparticle-bacterial interaction and suggests that the presence of filamentous morphs of B. subtilis may be due to physiological changes in some cells within the biofilm matrix. Non-motile, long cell chains have been reported previously in B. subtilis biofilms [44] and in many other bacteria including B. cereus, E. coli, L monocytogenes and Salmonella spp. [45]. Cell elongation/changes in morphology has been described as a survival mechanism to confer protection against stressful environmental situations [46, 47]. These morphotypes are regarded as viable since filamentation is a reversible stress response and the cells are able to divide and form new normal cells upon removal of the stress conditions [45]. According to scanning electron micrographs, IONs@APTES were seen concentrated within biofilms without the need of an external magnetic field (Figure 9f). Nanoparticles were also seen attached to the extracellular polymeric matrix which forms a complex network in biofilms (Figure 9f). As the majority of bacteria have negatively charged biofilm matrixes [48], positively charged IONs@APTES with high surface/volume ratio, which also disperse easily in the aqueous medium, can readily penetrate and bind to the biofilm matrix. Previous studies have also demonstrated that positively charged particles would more easily penetrate the negatively charged biofilms [34]. Apart from the charge, the size of IONs can also impact their diffusion through biofilms[34]. IONs@APTES significantly reduced the biofilm density as compared to the untreated cells (Figure 10). Despite the fact that IONs@APTES did not effectively kill biofilm bacteria in comparison to the untreated cells, CLSM analysis revealed that biofilm maximum depth, which is an indirect measure of the amount of B. subtilis biofilm biomass in the presence of
IONs@APTES, is 28.66 µm in depth which was significantly different from the depth of the biofilm in the absence of nanoparticles which was 44.60 µm (p<0.05) (Figure 11). Taken together, the results support the idea that IONs@APTES, with positive surface potential, hold great promise in controlling B. subtilis biofilm formation without compromising bacterial growth and viability.
Conclusions Growth and biofilm formation of B. subtilis has a profound effect on many bacterial bioprocesses. The present study was focused on immobilization of B. subtilis with naked and APTES coated IONs and evaluation of their effects on B. subtilis biofilm formation, growth and viability. In comparison to naked IONS, IONs@APTES offered a high density of surface functional groups of –NH3+ to IONs, promoting strong electrostatic interaction with negatively charged sites on the cell membranes allowing good decoration of cells. IONs@APTES have been demonstrated to be a promising approach in control of biofilm formation without the loss of B. subtilis cell viability compared to naked IONs. APTES coated magnetic nanoparticles, at appropriate concentrations, offer a considerable advantage for biofilm control while maintaining the growth and viability of bacterial cells when designing the next generation of bioprocesses intensification methods. Because of the overall significance of biofilm control, it is necessary to further study the spatial and temporal patterns of biofilm formation in the presence of IONs@APTES and the molecular basis of these interactions using an appropriate platform for biofilm experiments.
Ethical approval This study does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements This investigation was financially supported by The University of Waikato, New Zealand. The support given by Ms. Helen Turner, Dr. Barry O’Brian, Dr. Judith Burrows and Dr. Lisa Lee is highly appreciated. A special thanks to Dr. Andrew J. Spheres for the valuable suggestions in biofilm studies.
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Figure captions
Fig. 1 Transmission electron micrographs of a) naked IONs and b) IONs@APTES
Fig. 2 Fourier transform infrared spectroscopy (FTIR) spectra of a) naked IONs and b) IONs@APTES
Fig. 3 X—ray powder diffraction patterns of a) naked IONS and b) IONs@APTES
Fig. 4 Vibrating sample magnetometer (VSM) diagrams of a) naked IONs and b) IONs@APTES
Fig. 5 a crystal violet quantification of B. subtilis biofilm in the presence of IONs@APTES and naked IONs, b crystal violet stained biofilms using different IONs@APTES concentration (from left to right 0-400 µg/mL) and c crystal violet stained biofilms using different naked IONS concentrations (from left to right 0-400 µg/mL). Results are expressed as the absorbance measured at 550nm. Data are the means + standard error of 3 replicates. * p<0.05
Fig. 6 Quantification of B. subtilis biofilm pellicle biomass in the presence of IONs@APTES and naked IONs.The formation of a biofilm was measured after 60 hours using the pellicle biomass assay. Results are expressed as dry weight (g). Data are the means + standard error of 3 replicates. * p<0.05
Fig. 7 Population density of B. subtilis in the presence of IONs@APTES and naked IONs. Population density was measured by reading OD values at 600nm after 60 hours. Data are the means + standard error of 3 replicates. * p<0.05
Fig. 8 Viability of B. subtilis in the presence of IONs@APTES and naked IONs. Viability was assessed using LIVE/DEAD BacLight assay kit L7012 after 60 hours. Data are the means + standard error of 3 replicates. * p<0.05
Fig. 9 Scanning electron micrographs of B. subtilis growing in the presence of a IONs@APTES, b naked IONs, c control conditions, and filamentous morphs of B. subtilis growing d in the presence of IONs@APTES, e in the absence of nanoparticles, f B. subtilis biofilm matrix
Fig. 10 CLSM images of 60-h B. subtilis biofilm in the presence of 100µg/mL IONs@APTES (a and b) and in the absence of nanoparticles (c and d). Biofilms were stained with live/dead. Live cells are stained in green (a and c) dead cells stained in red (b and d)
Fig. 11 Biofilm maximum depth average obtained by CLSM for B. subtilis biofilm in the absence of nanoparticles and in the presence of 100 µg/mL IONs@APTES after 60 hours. *statistically different from control (untreated cells) (p< 0. 05)